We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1-38 kbp) and large DNA fragments (lambda DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas lambda DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.

Two types of low-voltage electroosmosis pumps were developed using microfabrication technology for usage in handy or stand-alone applications of the micrototal analysis systems (micro-TAS) and the lab-on-a-chip. This was done by making a thin (< 1 microm) region in the flow path and by only applying voltages near this thin region using electrodes inserted into the flow path. The inserted electrodes must be free from bubble formation and be gas-tight in order to avoid pressure leakage. For these electrodes, Ag/AgCl or a gel salt bridge was used. For patterning the gel on the chip, a hydrophilic photopolymerization gel and a photolithographic technique were optimized for producing a gel with higher electric conductivity and higher mechanical strength. For high flow rate application, wide (33.2 mm) and thin (400 nm) pumping channels were compacted into a 1 mm x 6 mm area by folding. This pump achieves an 800 Pa static pressure and a flow of 415 nL/min at 10 V. For high-pressure application, a pump was designed with the thin and thick regions in series and positive and negative electrodes were inserted between them alternatively. This pump could increase the pumping pressure without increasing the supply voltage. A pump with 10-stage connections generated a pressure of 25 kPa at 10 V.

A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.